Abstract
The pulmonary surfactant apoprotein with a reduced denatured molecular mass of 26-38 kDa (PSP-A) has recently been identified as an inhibitor of surfactant phospholipid secretion by isolated rat alveolar type II cells. We have investigated some of the structural determinants of PSP-A that are relevant to the inhibitory process. The PSP-A was isolated from rats given an intratracheal instillation of silica. The yield of PSP-A from silica-treated animals was 20-40-fold higher than that obtained from untreated animals. Reduction of PSP-A with 2-mercaptoethanol caused a reversible loss of biological activity that was restored by mild oxidation. Alkylation of the protein with excess iodoacetamide also led to inactivation, although titration with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that the protein initially contained no free sulfhydryl moieties. Neither alkylation nor reduction plus alkylation completely prevented the formation of oligomers as determined by gel permeation analysis. The apparent molecular mass of PSP-A at 4 degrees C in low ionic strength buffers was 1.6 megadaltons, and at 37 degrees C in normal ionic strength buffers was greater than 1.5 megadaltons. Removal of the oligosaccharide moiety with endoglycosidase F also had no effect upon biological activity. Five distinct monoclonal antibodies recognizing peptides epitopes on PSP-A were produced. All monoclonal antibodies exhibited similar affinity for PSP-A and recognized the delipidated and deglycosylated form. Four monoclonal antibodies reacted with epitopes on PSP-A that altered its function as an inhibitor. One monoclonal antibody was clearly ineffective at altering the activity of PSP-A. These results demonstrate that: 1) disulfide bonds are required for the activity of PSP-A, 2) disruption of disulfides does not prevent the formation of oligomeric forms of PSP-A, 3) the oligosaccharide moiety is not essential for biological activity, and 4) monoclonal antibodies can be used to map the epitopes responsible for biological activity.
Highlights
The pulmonary surfactant apoprotein witahreduced’ isa denatured molecular mass of 26-38 kDa (PSP-A) has family of glycoproteins that have monomeric molecular recently been identified as an inhibitor of surfactant weights of 26,000,32,000, and 38,000when examined by phospholipid secretion by isolated rat alveolar type I1 electrophoresis under reducing anddenaturing conditions
Pulmonary surfactant secretion is stimulated by P-adrenergic agents and TPA [17, 27, 28]
In this paper we have examined some of the structuraldeterminants of PSP-A that are required for this biological activity
Summary
Digestion, the incubation was carried out for an additional 16 h with Purified PSP-A fromNormal and Silica-treated Rats Znhibthe addition of fresh endoglycosidase F. The reaction mixture was applied to a Sephadex G-50 (Pharmacia) column (1.6 X 58 cm) to separate free oligosaccharide from the deglycosylated form of PSPA. Preparation of Monoclonal Antibodies against PSP-A-The hybridomatechnique was carriedoutusingthemethod ‘of Kohlerand. The purified PSP-A was emulsified in Freund’s complete adjuvant and injected into BALB/c mice subcutaneously. The monoclonal antibodies obtained were amplified by growing hybrid cells as ascites in pristane-treated BALB/c mice and purified usingtheProtein A-Sepharose CL-4B(Pharmacia). A and F, monoclonal antibodies wasidentified usingtheimmunoblotting molecular weight standards: B and D,PSP-A from normal rats; C method of Towbin et al [24]. PSP-A was separated by NaDodS04- and E , PSP-A from silica-treated rats
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